CHD7 and SOX2 act in a common gene regulatory network during mammalian semicircular canal and cochlear development.

Inner ear morphogenesis requires tightly regulated epigenetic and transcriptional control of gene expression. CHD7, an ATP-dependent chromodomain helicase DNA-binding protein, and SOX2, an SRY-related HMG box pioneer transcription factor, are known to contribute to vestibular and auditory system development, but their genetic interactions in the ear have not been explored. Here, we analyzed inner ear development and the transcriptional regulatory landscapes in mice with variable dosages of Chd7 and/or Sox2. We show that combined haploinsufficiency for Chd7 and Sox2 results in reduced otic cell proliferation, severe malformations of semicircular canals, and shortened cochleae with ectopic hair cells. Examination of mice with conditional, inducible Chd7 loss by Sox2CreER reveals a critical period (~E9.5) of susceptibility in the inner ear to combined Chd7 and Sox2 loss. Data from genome-wide RNA-sequencing and CUT&Tag studies in the otocyst show that CHD7 regulates Sox2 expression and acts early in a gene regulatory network to control expression of key otic patterning genes, including Pax2 and Otx2. CHD7 and SOX2 directly bind independently and cooperatively at transcription start sites and enhancers to regulate otic progenitor cell gene expression. Together, our findings reveal essential roles for Chd7 and Sox2 in early inner ear development and may be applicable for syndromic and other forms of hearing or balance disorders.

Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length.

Mutations in the chromatin remodeling factor CHD7 are the predominant cause of CHARGE syndrome, a congenital disorder that frequently includes ocular coloboma. Although CHD7 is known to be required for proper ocular morphogenesis, its role in retinal development has not been thoroughly investigated. Given that individuals with CHARGE syndrome can experience visual impairment even in the absence of coloboma, a better understanding of CHD7 function in the retina is needed. In this study, we characterized the expression pattern of Chd7 in the developing zebrafish and mouse retina and documented ocular and retinal phenotypes in Chd7 loss-of-function mutants. Zebrafish Chd7 was expressed throughout the retinal neuroepithelium when retinal progenitor cells were actively proliferating, and later in subsets of newly post-mitotic retinal cells. At stages of retinal development when most retinal cell types had terminally differentiated, Chd7 expression remained strong in the ganglion cell layer and in some cells in the inner nuclear layer. Intriguingly, strong expression of Chd7 was also observed in the outer nuclear layer where it was co-expressed with markers of post-mitotic cone and rod photoreceptors. Expression of mouse CHD7 displayed a similar pattern, including expression in the ganglion cells, subsets of inner nuclear layer cells, and in the distal outer nuclear layer as late as P15. Two different mutant chd7 zebrafish lines were characterized for ocular and retinal defects. These mutants displayed microphthalmia, reduced numbers of cone photoreceptors, and truncated rod and cone photoreceptor outer segments. Reduced cone photoreceptor number and abnormal outer segments were also observed in heterozygous Chd7 mutant mice. Taken together, our results in zebrafish and mouse reveal a conserved, previously undescribed role for Chd7 in retinal development and photoreceptor outer segment morphogenesis. Moreover, our work suggests an avenue of future investigation into the pathogenesis of visual system defects in CHARGE syndrome.

Chromatin remodeler CHD7 is critical for cochlear morphogenesis and neurosensory patterning.

Epigenetic regulation of gene transcription by chromatin remodeling proteins has recently emerged as an important contributing factor in inner ear development. Pathogenic variants in CHD7, the gene encoding Chromodomain Helicase DNA binding protein 7, cause CHARGE syndrome, which presents with malformations in the developing ear. Chd7 is broadly expressed in the developing mouse otocyst and mature auditory epithelium, yet the pathogenic effects of Chd7 loss in the cochlea are not well understood. Here we characterized cochlear epithelial phenotypes in mice with deletion of Chd7 throughout the otocyst (using Foxg1Cre/+ and Pax2Cre), in the otic mesenchyme (using TCre), in hair cells (using Atoh1Cre), in developing neuroblasts (using NgnCre), or in spiral ganglion neurons (using ShhCre/+). Pan-otic deletion of Chd7 resulted in shortened cochleae with aberrant projections and axonal looping, disorganized, supernumerary hair cells at the apical turn and a narrowed epithelium with missing hair cells in the middle region. Deletion of Chd7 in the otic mesenchyme had no effect on overall cochlear morphology. Loss of Chd7 in hair cells did not disrupt their formation or organization of the auditory epithelium. Similarly, absence of Chd7 in spiral ganglion neurons had no effect on axonal projections. In contrast, deletion of Chd7 in developing neuroblasts led to smaller spiral ganglia and disorganized cochlear neurites. Together, these observations reveal dosage-, tissue-, and time-sensitive cell autonomous roles for Chd7 in cochlear elongation and cochlear neuron organization, with minimal functions for Chd7 in hair cells. These studies provide novel information about roles for Chd7 in development of auditory neurons.

Genetic analysis of CHARGE syndrome identifies overlapping molecular biology.

PURPOSE: CHARGE syndrome is an autosomal-dominant, multiple congenital anomaly condition characterized by vision and hearing loss, congenital heart disease, and malformations of craniofacial and other structures. Pathogenic variants in CHD7, encoding adenosine triphosphate-dependent chromodomain helicase DNA binding protein 7, are present in the majority of affected individuals. However, no causal variant can be found in 5-30% (depending on the cohort) of individuals with a clinical diagnosis of CHARGE syndrome. METHODS: We performed whole-exome sequencing (WES) on 28 families from which at least one individual presented with features highly suggestive of CHARGE syndrome. RESULTS: Pathogenic variants in CHD7 were present in 15 of 28 individuals (53.6%), whereas 4 (14.3%) individuals had pathogenic variants in other genes (RERE, KMT2D, EP300, or PUF60). A variant of uncertain clinical significance in KDM6A was identified in one (3.5%) individual. The remaining eight (28.6%) individuals were not found to have pathogenic variants by WES. CONCLUSION: These results demonstrate that the phenotypic features of CHARGE syndrome overlap with multiple other rare single-gene syndromes. Additionally, they implicate a shared molecular pathology that disrupts epigenetic regulation of multiple-organ development.

CHD7 and retinoic acid signaling cooperate to regulate neural stem cell and inner ear development in mouse models of CHARGE syndrome.

CHARGE syndrome is a multiple congenital anomaly disorder that leads to life-threatening birth defects, such as choanal atresia and cardiac malformations as well as multiple sensory impairments, that affect hearing, vision, olfaction and balance. CHARGE is caused by heterozygous mutations in CHD7, which encodes an ATP-dependent chromatin remodeling enzyme. Identification of the mechanisms underlying neurological and sensory defects in CHARGE is a first step toward developing treatments for CHARGE individuals. Here, we used mouse models of Chd7 deficiency to explore the function of CHD7 in the development of the subventricular zone (SVZ) neural stem cell niche and inner ear, structures that are important for olfactory bulb neurogenesis and hearing and balance, respectively. We found that loss of Chd7 results in cell-autonomous proliferative, neurogenic and self-renewal defects in the perinatal and mature mouse SVZ stem cell niche. Modulation of retinoic acid (RA) signaling prevented in vivo inner ear and in vitro neural stem cell defects caused by Chd7 deficiency. Our findings demonstrate critical, cooperative roles for RA and CHD7 in SVZ neural stem cell function and inner ear development, suggesting that altered RA signaling may be an effective method for treating Chd7 deficiency.

Pleiotropic and isoform-specific functions for Pitx2 in superior colliculus and hypothalamic neuronal development.

Transcriptional regulation of gene expression during development is critical for proper neuronal differentiation and migration. Alternative splicing and differential isoform expression have been demonstrated for most mammalian genes, but their specific contributions to gene function are not well understood. In mice, the transcription factor gene Pitx2 is expressed as three different isoforms (PITX2A, PITX2B, and PITX2C) which have unique amino termini and common DNA binding homeodomains and carboxyl termini. The specific roles of these isoforms in neuronal development are not known. Here we report the onset of Pitx2ab and Pitx2c isoform-specific expression by E9.5 in the developing mouse brain. Using isoform-specific Pitx2 deletion mouse strains, we show that collicular neuron migration requires PITX2AB and that collicular GABAergic differentiation and targeting of hypothalamic projections require unique Pitx2 isoform dosage. These results provide insights into Pitx2 dosage and isoform-specific requirements underlying midbrain and hypothalamic development.

Disruption of RAB40AL function leads to Martin--Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder.

BACKGROUND AND AIM: Martin--Probst syndrome (MPS) is a rare X-linked disorder characterised by deafness, cognitive impairment, short stature and distinct craniofacial dysmorphisms, among other features. The authors sought to identify the causative mutation for MPS. METHODS AND RESULTS: Massively parallel sequencing in two affected, related male subjects with MPS identified a RAB40AL (also called RLGP) missense mutation (chrX:102,079,078-102,079,079AC→GA p.D59G; hg18). RAB40AL encodes a small Ras-like GTPase protein with one suppressor of cytokine signalling box. The p.D59G variant is located in a highly conserved region of the GTPase domain between ß-2 and ß-3 strands. Using RT-PCR, the authors show that RAB40AL is expressed in human fetal and adult brain and kidney, and adult lung, heart, liver and skeletal muscle. RAB40AL appears to be a primate innovation, with no orthologues found in mouse, Xenopus or zebrafish. Western analysis and fluorescence microscopy of GFP-tagged RAB40AL constructs from transiently transfected COS7 cells show that the D59G missense change renders RAB40AL unstable and disrupts its cytoplasmic localisation. CONCLUSIONS: This is the first study to show that mutation of RAB40AL is associated with a human disorder. Identification of RAB40AL as the gene mutated in MPS allows for further investigations into the molecular mechanism(s) of RAB40AL and its roles in diverse processes such as cognition, hearing and skeletal development.

Distinct populations of GABAergic neurons in mouse rhombomere 1 express but do not require the homeodomain transcription factor PITX2.

Hindbrain rhombomere 1 (r1) is located caudal to the isthmus, a critical organizer region, and rostral to rhombomere 2 in the developing mouse brain. Dorsal r1 gives rise to the cerebellum, locus coeruleus, and several brainstem nuclei, whereas cells from ventral r1 contribute to the trochlear and trigeminal nuclei as well as serotonergic and GABAergic neurons of the dorsal raphe. Recent studies have identified several molecular events controlling dorsal r1 development. In contrast, very little is known about ventral r1 gene expression and the genetic mechanisms regulating its formation. Neurons with distinct neurotransmitter phenotypes have been identified in ventral r1 including GABAergic, serotonergic, and cholinergic neurons. Here we show that PITX2 marks a distinct population of GABAergic neurons in mouse embryonic ventral r1. This population appears to retain its GABAergic identity even in the absence of PITX2. We provide a comprehensive map of markers that places these PITX2-positive GABAergic neurons in a region of r1 that intersects and is potentially in communication with the dorsal raphe.

A novel TaulacZ allele reveals a requirement for Pitx2 in formation of the mammillothalamic tract.

The hypothalamic mammillary region is critical for spatial memory and vestibular processing. Pitx2 encodes a paired-like transcription factor that is highly expressed in the developing mammillary region and is required for subthalamic nucleus formation. Here we analyzed a loss of function Pitx2-TaulacZ knock-in allele to study the effects of Pitx2 deficiency on neuronal projections in the embryonic mammillary region. Pitx2-expressing neurons contribute axons to principal mammillary, mammillotegmental and mammillotectal tracts. Embryos with Pitx2 deficiency exhibit axonal fibers in the principal mammillary tract that are improperly bundled and disorganized, yet project caudally toward the tectum and tegmentum. Embryos with Nestin-Cre mediated conditional Pitx2 deficiency exhibit truncated mammillothalamic tracts (mtt) that fail to elongate, and reduced Pax6-positive cells at the branching point of the principal mammillary and mtt. These data suggest that Pitx2 mediates cell-autonomous and nonautonomous guidance cues necessary for mammillary collaterals destined to project to the anterior thalamus.

GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function.

Pathogenic variants in GJB2, the gene encoding connexin 26, are the most common cause of autosomal-recessive hereditary deafness. Despite this high prevalence, pathogenic mechanisms leading to GJB2-related deafness are not well understood, and cures are absent. Humans with GJB2-related deafness retain at least some auditory hair cells and neurons, and their deafness is usually stable. In contrast, mice with conditional loss of Gjb2 in supporting cells exhibit extensive loss of hair cells and neurons and rapidly progress to profound deafness, precluding the application of therapies that require intact cochlear cells. In an attempt to design a less severe Gjb2 animal model, we generated mice with inducible Sox10iCre ERT2 -mediated loss of Gjb2. Tamoxifen injection led to reduced connexin 26 expression and impaired function, but cochlear hair cells and neurons survived for 2 months, allowing phenotypic rescue attempts within this time. AAV-mediated gene transfer of GJB2 in mature mutant ears did not demonstrate threshold improvement and in some animals exacerbated hearing loss and resulted in hair cell loss. We conclude that Sox10iCre ERT2 ;Gjb2 flox/flox mice are valuable for studying the biology of connexin 26 in the cochlea. In particular, these mice may be useful for evaluating gene therapy vectors and development of therapies for GJB2-related deafness.

CHD7 promotes neural progenitor differentiation in embryonic stem cells via altered chromatin accessibility and nascent gene expression.

CHARGE syndrome, a rare multiple congenital anomaly condition, is caused by haploinsufficiency of the chromatin remodeling protein gene CHD7 (Chromodomain helicase DNA binding protein 7). Brain abnormalities and intellectual disability are commonly observed in individuals with CHARGE, and neuronal differentiation is reduced in CHARGE patient-derived iPSCs and conditional knockout mouse brains. However, the mechanisms of CHD7 function in nervous system development are not well understood. In this study, we asked whether CHD7 promotes gene transcription in neural progenitor cells via changes in chromatin accessibility. We used Chd7 null embryonic stem cells (ESCs) derived from Chd7 mutant mouse blastocysts as a tool to investigate roles of CHD7 in neuronal and glial differentiation. Loss of Chd7 significantly reduced neuronal and glial differentiation. Sholl analysis showed that loss of Chd7 impaired neuronal complexity and neurite length in differentiated neurons. Genome-wide studies demonstrated that loss of Chd7 leads to modified chromatin accessibility (ATAC-seq) and differential nascent expression (Bru-Seq) of neural-specific genes. These results suggest that CHD7 acts preferentially to alter chromatin accessibility of key genes during the transition of NPCs to neurons to promote differentiation. Our results form a basis for understanding the cell stage-specific roles for CHD7-mediated chromatin remodeling during cell lineage acquisition.

Cre fate mapping reveals lineage specific defects in neuronal migration with loss of Pitx2 function in the developing mouse hypothalamus and subthalamic nucleus.

Establishment of neuronal diversity is a central topic in developmental neurobiology. Prior studies implicated Pitx2, a paired-like homeodomain transcription factor, in mouse subthalamic nucleus neuronal development, but precise stages of neuronal differentiation affected (migration, axon outgrowth, fate specification) and underlying mechanisms were unknown. Here we report lineage tracing experiments using Pitx2(cre/+), Pitx2(cre/null), and conditional nuclear lacZ reporter mice to track embryonic Pitx2 expressing neurons. Migration of subthalamic nucleus and hypothalamic neurons was severely arrested in Pitx2(cre/null) embryos, and subclasses of subthalamic nucleus neurons identified by Lmx1b, Foxp1, and Foxp2-gene expression revealed differing sensitivities to Pitx2 dosage. Interestingly, embryonic subthalamic nucleus development was unaffected in Lmx1b null mice, suggesting that Pitx2 and Lmx1b act via independent genetic pathways. These data provide the first direct evidence for Pitx2-dependent neuronal migration in the developing hypothalamus, and demonstrate that complex transcriptional networks regulate regional specialization of distinct hypothalamic and subthalamic nucleus neurons.

CHD7 represses the retinoic acid synthesis enzyme ALDH1A3 during inner ear development.

CHD7, an ATP-dependent chromatin remodeler, is disrupted in CHARGE syndrome, an autosomal dominant disorder characterized by variably penetrant abnormalities in craniofacial, cardiac, and nervous system tissues. The inner ear is uniquely sensitive to CHD7 levels and is the most commonly affected organ in individuals with CHARGE. Interestingly, upregulation or downregulation of retinoic acid (RA) signaling during embryogenesis also leads to developmental defects similar to those in CHARGE syndrome, suggesting that CHD7 and RA may have common target genes or signaling pathways. Here, we tested three separate potential mechanisms for CHD7 and RA interaction: (a) direct binding of CHD7 with RA receptors, (b) regulation of CHD7 levels by RA, and (c) CHD7 binding and regulation of RA-related genes. We show that CHD7 directly regulates expression of Aldh1a3, the gene encoding the RA synthetic enzyme ALDH1A3 and that loss of Aldh1a3 partially rescues Chd7 mutant mouse inner ear defects. Together, these studies indicate that ALDH1A3 acts with CHD7 in a common genetic pathway to regulate inner ear development, providing insights into how CHD7 and RA regulate gene expression and morphogenesis in the developing embryo.

Characterization of progenitor domains in the developing mouse thalamus.

To understand the molecular basis of the specification of thalamic nuclei, we analyzed the expression patterns of various transcription factors and defined progenitor cell populations in the embryonic mouse thalamus. We show that the basic helix-loop-helix (bHLH) transcription factor Olig3 is expressed in the entire thalamic ventricular zone and the zona limitans intrathalamica (ZLI). Next, we define two distinct progenitor domains within the thalamus, which we name pTH-R and pTH-C, located caudal to the ZLI. pTH-R is immediately caudal to the ZLI and expresses Nkx2.2, Mash1, and Olig3. pTH-C is caudal to pTH-R and expresses Ngn1, Ngn2, and Olig3. Short-term lineage analysis of Olig3-, Mash1-, Ngn1-, and Ngn2-expressing progenitor cells as well as tracing the Pitx2 cell lineage suggests that pTH-C is the only major source of thalamic nuclei containing neurons that project to the cerebral cortex, whereas pTH-R and ZLI are likely to produce distinct postmitotic populations outside of the cortex-projecting part of the thalamus. To determine if pTH-C is composed of subdomains, we characterized expression of the homeodomain protein Dbx1 and the bHLH protein Olig2. We show that Dbx1 is expressed in caudodorsal-high to rostroventral-low gradient within pTH-C. Analysis of heterozygous Dbx1(nlslacZ) knockin mice demonstrated that Dbx1-expressing progenitors preferentially give rise to caudodorsal thalamic nuclei. Olig2 is expressed in an opposite gradient within pTH-C to that of Dbx1. These results establish the molecular heterogeneity within the progenitor cells of the thalamus, and suggest that such heterogeneity contributes to the specification of thalamic nuclei.

Genetic specification of left-right asymmetry in the diaphragm muscles and their motor innervation.

The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L-R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.

Nestin-lineage cells contribute to the microvasculature but not endocrine cells of the islet.

To clarify the lineage relationship between cells that express the neural stem cell marker nestin and endocrine cells of the pancreas, we analyzed offspring of a cross between mice carrying a nestin promoter/enhancer-driven cre-recombinase (Nestin-cre) and C57BL/6J-Gtrosa26(tm1Sor) mice that carry a loxP-disrupted beta-galactosidase gene (Rosa26). In nestin-cre(+/tg);R26R(loxP/+) embryos, cre-recombinase was detected in association with nestin-positive cells in the pancreatic mesenchyme with some of the nestin-positive cells lining vascular channels. In postnatal mice, pancreatic beta-galactosidase expression was restricted to vascular endothelial cells of the islet and a subset of cells in the muscularis of arteries in a distribution identical to endogenous nestin expression. Ex vivo explants of mouse pancreatic ducts grew dense cultures that costained for nestin and beta-galactosidase, demonstrating recombination in vitro. The cultures could be differentiated into complex stereotypic structures that contain nestin- and insulin-expressing cells. Nestin-cre(+/tg);R26R(loxP/+)-derived duct cultures showed that insulin-positive cells were negative for beta-galactosidase. These results indicate that both in vivo and in vitro pancreatic endocrine cells arise independently of nestin-positive precursors. The apparent vascular nature of the nestin-positive cell population and the close association with endocrine cells suggest that nestin-positive cells play an important role in the growth and maintenance of the islet.

Nestin-Cre mediated deletion of Pitx2 in the mouse.

Nestin-Cre mice are widely used to generate gene deletions in the developing brain. Surprisingly, fewNestin-Cre lines have been characterized for their temporal and brain region-specific recombination. In addition, some Nestin-Cre lines express Cre outside the central nervous system, making it difficult to choose appropriate lines for targeting genes with brain region-restricted expression. Here we describe the properties of a Nestin-Cre transgenic line and its use for conditional deletions of Pitx2, a paired-like homeodomain transcription factor. We report that Nestin-Cre conditional Pitx2 mutant mice have ocular and craniofacial defects consistent with the role of human PITX2 in Rieger syndrome. Conditional mutants exhibit defects in midbrain neuronal development similar to those in Pitx2 homozygous null embryos, but lack the abnormalities in subthalamic nucleus neurons that occur with complete loss of Pitx2 function. These data indicate that normal differentiation of midbrain neurons depends upon adequate Pitx2 function during the period of active neurogenesis.

The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels.

The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtAC) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtAC, we generated a temperature-sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtAC is important for 50S assembly and/or stability. Surprisingly, at the non-permissive temperature, G80E cells rapidly lose viability and yet do not display an additional ribosome defect. Thus, the essential nature of the cgtAC gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1-S arrest. Therefore, CgtAC is necessary for DNA replication and progression through the cell cycle.

Pitx2 distinguishes subtypes of terminally differentiated neurons in the developing mouse neuroepithelium.

Pitx2, a homeodomain transcription factor, is essential for normal development of pituitary, eyes, heart, and teeth. In the developing mouse brain, Pitx2 (Rieg, Ptx2, Otlx2, Brx1) mRNA is expressed in discrete regions of the diencephalon, mesencephalon, and rhombencephalon. While prior reports have provided an overview of the temporal and regional specificity of Pitx2 mRNA expression in the brain, the precise cell types that express PITX2 are not known. In this study, we analyzed Pitx2 mRNA and PITX2 protein expression in individual cells of the developing e10.5-e14.5 mouse CNS using multiple markers of cellular proliferation and differentiation. We identified Pitx2 expression in nestin-positive neural progenitors and in postmitotic, developing neurons. In the diencephalon, PITX2 is expressed in neurons of the zona limitans intrathalamica and mammillary region and in gamma-aminobutyric acid (GABA)-producing neurons of the zona incerta. In the mesencephalon, PITX2-labeled nuclei also appear in differentiated neurons, some of which are GABAergic and destined to occupy superior colliculus. Our results suggest that PITX2 expression in postmitotic neurons may contribute to development of GABAergic and other differentiated neuronal phenotypes.

PITX2 is required for normal development of neurons in the mouse subthalamic nucleus and midbrain.

Pitx2, a homeodomain transcription factor, is essential for normal development of the pituitary gland, craniofacial region, eyes, heart, abdominal viscera, and limbs. Complete loss of Pitx2 in mice (Pitx2(-/-)) results in embryonic lethality by approximately e15 due to cardiac defects, whereas embryos with partial loss of function (Pitx2(neo/-) or Pitx2(neo/neo)) survive until later in development (e17-e19). Pitx2 is expressed in discrete populations of postmitotic neurons in the mouse brain, but its role in mammalian central nervous system (CNS) development is not known. We undertook an analysis of Pitx2-deficient embryos to determine whether loss of Pitx2 affects CNS development. The CNS is normal in hypomorphic e16.5 Pitx2(neo/-) and e18.5 Pitx2(neo/neo) embryos, with no evidence of midline or other defects. Midgestation (e10.5) Pitx2(-/-) embryos have normally formed neural tube structures and cerebral vesicles, whereas older (e14.5) Pitx2(-/-) embryos exhibit loss of gene expression and axonal projections in the subthalamic nucleus (a group of cells in the ventrolateral thalamus) and in the developing superior colliculus of dorsal midbrain. Our results suggest a role for Pitx2 in regulating regionally specific terminal neuronal differentiation in the developing ventrolateral thalamus and midbrain.